Until recently the localization of intravascular drugs in body tissues has depended on chemical partitioning across microvascular barriers into the tissue compartments of multiple body organs. This resulted in only 0.01% to 0.001% of the injected dose actually reaching the intended targets. Approximately 20 years ago, drugs were entrapped in liposomes and microspheres. This modified the initial biodistributions and redirected them to phagocytes in the reticuloendothelial organs: liver, spleen and bone marrow.
In 1978, the present inventor and coworkers (Widder, et al., Proc. Am. Assn. Cancer Res., V. 19, p 17 (1978)) developed a means to co-entrap drug plus magnetite in microspheres which could be injected intravenously and localized magnetically in the tissue compartments of nonreticuloendothelial target organs (e.g., lung and brain). Magnetic capture was accomplished by selective dragging of the particles through the vascular endothelium into normal tissues and tissue tumors positioned adjacent to an extracorporeal magnet of sufficient strength (0.5 to 0.8 Tesla) and gradient (0.1 Tesla/mm). Although this technique was highly efficient and deposited between 25% and 50% of an injected dose in the desired target tissue, it was also a very complicated approach which had the following major disadvantages: 1) restriction of use to specialized medical centers; 2) permanent disposition of magnetite in target tissue; 3) focal overdosing of drug due to inhomogeneity of the capturing magnetic field; and 4) application to a very limited number of therapeutic agents. In the process of studying magnetic targeting, however, it was learned that slow (controlled) release of toxic drugs from entrapment-type carriers (microspheres) protected the normal cells within the local tissue environment from drug toxicity and still gave effective treatment of tumor cells and microorganisms.
When monoclonal antibodies became generally available for animal and clinical research, it was hoped that antibody-drug conjugates would limit the biodistribution of toxic agents and cause them to become deposited in foci of disease (tumors and infections) which were located across the microvascular barrier within target tissues. Unfortunately, most monoclonal antibodies were (and are still) obtained from mice, making them immunologically foreign to human recipients. Conjugation of drugs at therapeutically relevant substitution ratios makes the monoclonal antibody derivatives even more foreign and impairs their binding specificities. Hence, antibody-drug conjugates are cleared substantially by the liver, as are liposomes. Importantly, their localization in most solid tumors is even further impaired by the presence of a partially intact microvascular barrier which separates the tumor tissue (interstitium) from the bloodstream. This allows only about 1% to 7% (at best) of the injected dose to reach nonreticuloendothelial targets. Selected lymphomas and leukemias provide exceptions to this rule because of a greater natural breakdown of this vascular barrier. However, for the vast majority of solid tumors and infections, a general-purpose method is still needed to deliver drugs efficiently across microvascular barriers in a depot (controlled release) form.
Such a form of drugs is necessary in order to protect the normal vascular endothelium, organs and tissue cells from the toxic effects of drugs, protect the drug from endothelial and tissue metabolism during transit and make the drug bioavailable at a controlled therapeutic rate selectively within the target tissues and tissue lesions, including solid tumors.
Active endothelial transport has been demonstrated for small molecules (e.g., glucose and insulin), however, no studies other those that of the present inventor have shown such transport for larger molecules or molecules carried in a cargo format. Present examples show that transendothelial migration of macromolecular conjugates and noncovalent paired-ion formulations of drugs and diagnostic agents with sulfated glycosaminoglycan, having a combined size of between about 8,000 daltons and about 500 nanometers, are accelerated by the inclusion of sulfated glycosaminoglycans, and in particular, dermatan sulfates, which bind multiply to receptors or antigens which are either synthesized by disease-induced endothelium or are synthesized at other sites, but become selectively bound to the induced endothelial receptors at sites of disease. (Ranney, Biochem. Pharmacology, V. 35, No. 7, pp. 1063-1069 (1986)).
The present invention describes improved novel compositions, carriers, agents and methods of in vivo use which give improved selectivity, efficacy, uptake mechanism and kinetic-spatial profiles at sites of disease. It further describes compositions, agents and methods of use for improved selectivity, sensitivity, uptake mechanism and kinetic-spatial profiles of in vivo selective drug localization, accumulation and action at sites of disease, including but not limited to solid tumors. Novel compositions are prepared by (a) unique non-covalent chemical binding, further enhanced by (b) physical stabilization. Other compositions are prepared by covalent chemical binding. Binding is of cationic or chemically basic metal chelators to carriers comprising anionic or chemically acidic saccharides, sulfatoids and glycosaminoglycans, typically and advantageously of a hydrophilic or essentially completely hydrophilic nature. Binding of the active and carrier may also be by a combination of non-covalent, physical, and covalent means. Non-covalent binding can be carried out by means including but not limited to admixing cationic or basic drugs and metal chelates at appropriate ratios with anionic or acidic saccharide carriers, thereby forming strong solution-state and dry-state paired-ion complexes and salts, respectively, based principally on electrostatic binding of cationic (basic) group or groups of the metal chelator to anionic (acidic) group or groups of the acidic carrier. Such binding may be further stabilized by hydrogen bonds and physical factors, including but not limited to concentration, viscosity, and various means of drying, including lyophilization.
Carrier substances useful in this invention may include, but are not limited to natural and synthetic, native and modified, anionic or acidic saccharides, disaccharides, oligosaccharides, polysaccharides and glycosaminoglycans (GAGs) and in particular, dermatan sulfates. It will be apparent to those skilled in the art that a wide variety of additional biologically compatible, water-soluble and water dispersable, anionic carrier substances can also be used. Due to an absence of water-diffusion barriers, favorable initial biodistribution and multivalent site-binding properties, oligomeric and polymeric, hydrophilic and substantially completely hydrophilic carrier substances are included among the preferred carriers for agents to be used for treating tumors, cardiovascular infarcts and other types of local disease. However, it will be apparent to those skilled in the art that amphoteric and hydrophobic carriers may be favored for certain therapeutic applications. Drugs and metal chelators most useful in this invention include those which contain cationic, basic and basic-amine groups for binding to the carrier, and which are effective to treat local disease conditions either directly or indirectly, including by chelation of metals and metal ions, transition elements and ions, and lanthanide series elements and ions. It will be apparent to those skilled in the art that essentially any single atomic element or ion amenable to chelation by a cationic, basic and amine-containing chelator, may also be useful in this invention.
For purposes of this invention, a cationic or basic metal chelator is defined and further distinguished from a metal-ion complex as follows: a cationic or basic metal chelator comprises an organic, covalent, bridge-ligand molecule, capable of partly or entirely surrounding a single metal atom or ion, wherein the resulting formation constant of chelator for appropriate metal or ion is at least about 10.sup.14. A chelator is further defined as cationic or basic if it or its functional group or groups which confer the cationic or basic property, and which include but are not limited to an amine or amines, is (are) completely or essentially completely electrophilic, positively charged or protonated at a typical pH employed for formulation. A formulation pH is characteristically selected to closely bracket the range of physiologic pH present in mammalian vertebrates. This typically includes, but is not limited to a pH in the range of pH 5 to 8. Amines may include primary, secondary, tertiary or quaternary amines or combinations thereof on the metal chelator. Herein, and as specified, a hydrophilic carrier is defined as a substance which is water soluble, partitions into the water phase of aqueous-organic solvent mixtures, or forms a translucent aqueous solution, complex, aggregate, or particulate dispersion under the conditions employed for formulation. A carrier is further defined as being anionic or acidic if it is completely or nearly completely nucleophilic, or if its functional group or groups are capable of interacting with cationic, basic or amine metal chelators, is (are) completely or nearly completely negatively charged, anionic or ionized at the pH employed for formulation. Such anionic and acidic groups include, but are not limited to sulfates, phosphates and carboxylates, or combinations thereof on the carrier.
Novel agent compositions include, but are not limited to the classes of cationic or basic, typically basic-amine metal chelator actives, or metal chelator actives including the chelated metal or metal ion, wherein these actives are further bound to anionic and acidic carriers comprising natural or synthetic carriers, including but not limited to hydrophilic anionic or acidic, natural or synthetic, native, modified, derivatized and fragmented, anionic or acidic saccharides, oligosaccharides, polysaccharides, sulfatoids, and glycosaminoglycans (GAGs).
Anionic and acidic saccharide and glycosaminoglycan carriers may contain monomeric units comprising glucose, glucuronic acid, iduronic acid, glucosamine, galactose, galactosamine, xylose, mannose, fucose, sialic acid, pentose, and other naturally occurring, semi-synthetic or synthetic monosaccharides or chemical derivatives thereof, comprising amine, sulfate, carboxylate, sialyl, phosphate, hydroxyl or other side groups. Glycosaminoglycans (GAGs) comprise essentially the carbohydrate portions of cell-surface and tissue matrix proteoglycans. They are derived from naturally occurring proteoglycans by chemical separation and extraction; and in certain instances, by enzymatic means [Lindahl et al. (1978), incorporated herein by reference]. They include, but are not limited to those of the following types: heparin, heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, syndecan, and hyaluronate, and over-sulfated, hyper-sulfated, and other chemical derivatives thereof, as described further below.
The strongly acidic, sulfated glycosaminoglycans include all of those classes listed just above, except for hyaluronate, which contains only the more weakly acidic carboxylate groups and not sulfate groups. Natural sources of glycosaminoglycans include, but are not limited to: pig and beef intestinal mucosa, lung, spleen, pancreas, and a variety of other solid and parenchymal organs and tissues.
Sulfatoids comprise a second class of sulfated saccharide substances which are derived principally but not exclusively from bacterial and non-mammalian sources. Sulfatoids are typically of shorter chain length and lower molecular weight than glycosaminoglycans, but may be synthetically modified to give (a) longer chain lengths, (b) increased sulfation per unit saccharide, (c) various other chemical side groups, or (d) other properties favorable to the desired ligand-binding property and site-selective binding, uptake and accumulation property (or properties) in vivo. Sucrose and other short-chain oligosaccharides may be obtained from natural and synthetic sources.
These oligosaccharides can be rendered anionic or acidic by chemical or enzymatic derivatization with carboxylate, phosphate, sulfate or silyl side groups, or combinations thereof, at substitution ratios of up to about eight anionic or acidic substituent groups per disaccharide unit. Modified glycosaminoglycans may be derived from any of the types and sources of native glycosaminoglycans described above, and include: (1) glycosaminoglycan fragments, further defined as glycosaminoglycans with chain lengths made shorter than the parental material as isolated directly from natural sources by standard ion-exchange separation and solvent fractionation methods; (2) glycosaminoglycans chemically modified to decrease their anticoagulant activities, thereby giving "non-anticoagulant" (NAC) GAGs, prepared typically but not exclusively by (a) periodate oxidation followed by borohydride reduction; (b) partial or complete desulfation; and (c) formation of non-covalent divalent or trivalent counterion salts, principally including but not limited to salts of the more highly acidic sulfate functional groups, with principally but not exclusively: calcium, magnesium, manganese, iron, gadolinium and aluminum ions.
For purposes of this invention, a special class of such solution complexes and salts includes those strong complexes and salts formed by electrostatic or paired-ion association between the acidic or sulfate groups of acidic saccharide or glycosaminoglycan carrier, and the basic or cationic group or groups of the metal chelator or metal chelator including metal, as described above. Derivatized acidic saccharides and glycosaminoglycans are typically prepared by derivatization of various chemical side groups to various sites on the saccharide units. This may be performed by chemical or enzymatic means.
Enzymatic means are used in certain instances where highly selective derivatization is desired. Resulting chemical and enzymatic derivatives include, but are not limited to acidic saccharides and glycosaminoglycans derivatized by: (1) esterification of (a) carboxylate groups, (b) hydroxyl groups, and (c) sulfate groups; (2) oversulfation by nonselective chemical or selective enzymatic means; (3) acetylation, and (4) formation of various other ligand derivatives, including but not limited to (a) addition of sialyl side groups, (b) addition of fucosyl side groups, and (c) treatment with various carbodiimide, anhydride and isothiocyanate linking groups, and (d) addition of various other ligands.
If and when present, sulfate and sialyl side groups may be present at any compatible position of saccharide monomer, and on any compatible position of glycosaminoglycan monomers [Lindahl et al. (1978), incorporated herein by reference]. Certain of the resulting derivatized acidic saccharides and glycosaminoglycans may have desired alterations of anticoagulant activities, site-localization patterns, clearance and other biological properties. As one example of this relationship between certain classes of glycosaminoglycans and biological properties, dermatan sulfates with a native sulfate/carboxylate ratio preferably in the range of from 0.7:1 to 1.8:1, more preferably between 0.9:1 and 1.5:1 and typically 1:1, are reported to have relatively low binding to normal endothelial cells, avoid displacement of endogenous heparan sulfate from endothelial-cell surfaces, have relatively high selectivity to induced endothelia at sites of disease, including thrombus, and have rapid plasma clearance, principally by the renal route; whereas heparins and oversulfated dermatan sulfates with higher sulfate/carboxylate ratios of between 2:1 and 3.7:1, are reported to have relatively higher binding for both normal and induced endothelia, to displace relatively more endogenous endothelial heparan sulfate, and to clear more slowly than dermatans [Boneu et al. (1992), incorporated herein by reference].
As newly described and used in the present invention, the dermatan sulfate class of glycosaminoglycans, and especially the new special class of dermatan sulfates which contain selectively oversulfated oligosaccharide sequences, have the further unique advantages of higher potency combined with very low toxicity as carrier substances of associated or bound actives (i.e., dermatan sulfate-actives, DS-actives). This is related to their (a) relatively low sulfate/carboxylate ratios which range between 0.7:1 and 1.8:1, most preferably lying between 0.9:1 and 1.5:1, and most typically being 1:1; (b) very low anticoagulant activities--related to very low factor Xa and USP heparin activity plus negligible binding to antithrombin III; (c) very low or absent platelet-aggregating, and hence thrombocytopenia-inducing properties--related to their relatively low SO.sub.3 --/COO-ratios in combination with a modal molecular weight of less than about 45,000 daltons and preferably less than about 25,000 daltons; (d) essentially complete absence of in vivo metabolism; and (e) very rapid blood and body clearance, all as further described below. These properties result in an extremely high in vivo safety profile with an absence of bleeding, metabolism and in vivo residua in normal tissues and organs. These properties and their resulting safety profiles clearly distinguish the dermatan sulfates from all other classes of glycosaminoglycans (GAGs) and other classes of acidic saccharides, oligosaccharides, polysaccharides and sulfatoid substances (taken together, comprising acidic and anionic saccharide substances), and they provide uniquely surprising and unexpected advantages for dermatan sulfates over these other classes of acidic and anionic saccharides. Most particularly, the dermatan sulfates show these surprising and unexpected advantages over other glycosaminoglycan polysulfates, with SO.sub.3 --/COO-- ratios in the range of between 2:1 and 3.7:1 and sulfur contents of greater than or equal to 10% (weight basis--indicative of their much higher sulfate contents). Also, most particularly, the new special class of dermatan sulfates (as described at length below), which is enriched for selectively oversulfated oligosaccharide sequences without comprising oversulfated or polysulfated molecules overall throughout the entire chain length (the latter being characterized by SO.sub.3 --/COO-- ratios greater than or equal to 2.0:1 and sulfur contents greater than or equal 10%), have the further surprising and unexpected advantage of more strongly binding to the selectively induced receptors of endothelium, tissue matrix and target-cells at sites of disease (including tumors) by means of the complementary, selectively oversulfated oligosaccharide sequences of these new special dermatan sulfates. Hence, these new special dermatan sulfates exhibit surprisingly and unexpectedly more potent site localization and site-targeting potencies than would otherwise be expected based on their moderately low overall SO.sub.3 --/COO-- ratio and sulfation and on their related extremely low cellular and systemic toxicity properties and side-effect profiles.
In a special case unique to the present invention, derivatization of the acidic saccharide and glycosaminoglycan carriers may be accompanied by the basic metal chelator itself. Although the general classes of carriers described above are particularly suitable to the present invention, it will be apparent to those skilled in the art that a wide variety of additional native, derivatized and otherwise modified carriers and physical formulations thereof, may be particularly suitable for various applications of this invention. As one representative example, the source and type of glycosaminoglycans, its chain length and sulfate/carboxylate ratio can be optimized to (1) provide optimal formulation characteristics in combination with different small and macromolecular diagnostic agents and drugs; (2) modulate carrier localization on diseased versus normal endothelium; (3) minimize dose-related side effects; (4) optimize clearance rates and routes of the carrier and bound diagnostic and therapeutic actives.
Non-covalent formulations of active and carrier afford markedly higher active-to-carrier ratios than those possible for covalent chemical conjugates. In the present invention, non-covalent binding affords a minimum of 15% active to total agent by weight [active/(active+carrier), w/w]; typically greater than about 30% (w/w); preferably at least about 50% (w/w); and frequently between about 70-99% (w/w). Covalent binding characteristically limits the percent active to (a) less than about 12% for non-protein small and polymeric carriers, (b) less than about 7% for peptide and protein carriers, including antibodies, and (c) less than about 0.5-2.0% for antibody fragments. This limitation is based on the number of functional groups available on carrier molecules which are useful in agent formulation and in vivo site localization.
It will be apparent to those skilled in the art that covalent active-carrier agent compositions of low substitution ratio may be useful for certain in vivo applications of typically narrow range, and that non-covalent active-carrier agent compositions of high substitution ratio may be useful for other in vivo applications of typically broader range. Generally, but not exclusively, non-covalent agents may be particularly useful for the majority of diagnostic imaging applications and for most therapeutic applications, wherein high total-body and site-localized doses are needed, and rapid clearance of the non-localized fraction of administered agent is desired in order to accelerate plasma clearance and to achieve low background levels for purposes of maximizing image contrast and minimizing systemic drug toxicity.
These properties of the present formulations represent additional substantial improvements over prior, non-selective and covalently conjugated active-carrier agents. The resulting agents are broadly useful for: (a) site-selective drug localization, including tumors, infections and cardiovascular disease with an acute endothelial induction; (b) MRI contrast and spectral enhancement, Ultrasound contrast enhancement, and X-Ray contrast enhancement, where relatively high administered doses may be favored or required; (c) Nuclear Medical or Radionuclide imaging and therapy, where enhanced clearance of the non-targeted dose may be favored or required: and (d) certain high-dose, extended-release or sustained-effect therapy may be favored or required. Such therapeutic agents include but are not limited to those useful at a broad variety of organ sites and medical indications, for the treatment of: (a) acute vascular ischemia, acute infarct, acute vascular damage, shock, hypotension, restenosis, tumors and tumor angiogenesis and parenchymal-cell or other pathological proliferations; and (b) the following classes of disease: vascular, parenchymal, mesenchymal, endothelial, smooth muscle, striated muscle, adventitial, immune, inflammatory, bacterial, fungal, viral, degenerative, neoplastic, genetic and enzymatic.
MRI contrast enhancement and drug therapy are important indications for which high payload and controlled release of active agents are important unique advantages in addition to site selective localization (see below).
For purposes of this invention, potentially therapeutic metal ions generally useful for trans chelation at sites of disease may include divalent and trivalent cations selected from the group consisting of: iron, manganese, chromium, copper, aluminum, nickel, gallium, indium, gadolinium, erbium, europium, dysprosium and holmium. Chelated metal ions generally useful for radionuclide imaging and compositions and uses, and in radiotherapeutic compositions and uses, may include metals selected from the group consisting of: phosphorous, sulfur, gallium, iodine, germanium, cobalt, calcium, rubidium, yttrium, technetium, ruthenium, rhenium, indium, tin, iridium, platinum, thallium, strontium and samarium. Metal ions useful in neutron-capture radiation therapy may include boron and others with large nuclear cross sections. Metal ions useful in Ultrasound contrast and X-Ray contrast compositions and uses may, provided they achieve adequate site concentrations, include any of the metal ions listed above, and in particular, may include metal ions of atomic number at least equal to that of iron.
For purposes of this invention, agents for therapeutic composition and uses in chelating internal body iron, copper or both, in order to make these metals unavailable locally (1) which are typically required for neovascularization, or (2) which cause and amplify local tissue injury [Levine (1993), incorporated herein by reference], include the carrier with basic metal chelator in one or both of the following forms: (a) carrier plus chelator without metal ion; and (b) carrier plus chelator with metal ion added and chelated in the composition at a formation constant lower or equal to that of the internal body metal which is to be chelated by metal ion exchange into the respective basic metal chelator of the composition (see below). Such weakly chelated metal ions of the composition may include one selected from the group consisting of: calcium, manganese, magnesium, chromium, copper, zinc, nickel, iron, gallium, indium, aluminum, cobalt, gadolinium or other exchangeable ion. Metal ions useful for inclusion in compositions for other therapeutic uses may include the divalent and trivalent cations selected from the group consisting of magnesium, manganese, chromium, zinc and calcium, iron, copper and aluminum. It will be obvious to those skilled in the art that various ones of the preceding metal ions can be used in combination with basic metal chelators, for alternative indications than those specified above, and that metal ions other than those listed above may, under certain conditions, be useful in the uses and indications listed above.
The compositions described in this invention give surprising and unexpected improvements of performance and use which include:
(1) retained high association of active plus carrier during in vitro dialysis and in vivo targeting; PA1 (2) selective binding of the active plus carrier to induced endothelia at sites of disease; PA1 (3) following intravenous administration, very rapid (2-7 min) localization at the diseased site, due to rapid selective endothelial binding, envelopment and extravasation of the carrier plus metal chelator across disease-induced endothelia (including histologically non-porous endothelia); PA1 (4) widespread uptake throughout the diseased tissue site; PA1 (5) sustained retention (multiple hours to days) within the diseased site in combination with PA1 (6) rapid plasma clearance (minutes) of the non-targeted fraction; PA1 (7) moderately slower, polymeric backdiffusion rates into the plasma, affording prolonged disease-site retention; PA1 (8) capacity to selectively treat and image solid tumors or acute vascular and myocardial infarcts at body sites, as well as at brain and central nervous system sites, with substantially improved selectivity and sensitivity, including small tumor metastases, in liver, lung and other organ sites. PA1 1. Simple formulations of active and carrier; PA1 2. Stabilization of diagnostic and therapeutic actives on the shelf and during plasma transit; PA1 3. Rapid site localization and sustained site retention; PA1 4. Rapid clearance of the non-targeted fraction; PA1 5. Availability of low toxicity carbohydrate and glycosaminoglycan carriers from natural sources and, where needed, modification or derivatization by straightforward synthetic means. PA1 1. Local tissue diseases induce local cytokines and mediators, as described above. In particular, it is reported recently that the cytokine, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), is selectively induced by many or most tumors of human and animal origin [Senger et al. (1994), incorporated by reference herein] and is a 34-42 kDa heparin-binding and GAG-binding glycoprotein that acts directly on endothelial cells by way of specific endothelial receptors [Jakeman et al. (1993), incorporated by reference herein], to cause endothelial activation and induce additional new endothelial receptors which can bind GAGs (see below). VEGF/VPF is a chemically basic growth factor which is quite highly selective for endothelial cells versus fibroblasts and other cell types [Senger et al. (1994); Nicosia et al. (1994), incorporated by reference herein]. It appears to be a key growth factor for stimulating the long-term endothelial angiogenesis in many or most human and animal tumors, and in AIDS-associated Kaposi's sarcoma [Connolly et al. (1989); Weindel et al. (1992), both incorporated by reference herein]. In certain instances, VEGF/VPF may also be important for the more transient and anatomically restricted angiogenic processes of wound healing and vascular restenosis [Senger et al. (1994); Miller et al. (1994); Nicosia et al. (1994); Berse et al. (1992), all incorporated by reference herein]. VEGF/VPF and platelet-derived growth factor, PDGF-BB, are reported recently to be the only species of the group of basic, GAG-binding growth factors which have significant angiogenic potency in vitro, i.e., ones which are directly active in the absence of in vivo cofactors [Nicosia et al. (1994), incorporated by reference herein]. The effects of VEGF/VPF are inhibited by antibodies directed against certain peptides on the external surface of the molecule [Sioussat et al. (1993), incorporated by reference herein], and importantly, such inhibition suppresses the growth of animal tumors in vivo [Kim et al. (1993), incorporated by reference herein]. Hence, VEGF/VPF both provides and induces receptor targets for binding of GAG carrier substances in tumors and potentially in other pathologic lesions. PA1 2. These cytokines and mediators induce tissue chemoattractants, including VEGF/VPF, MCP (Yamashiro et al., 1994) and IL-8, which comprise a family of arginine-rich, 8Kd, heparin-binding proteins reported to bind GAGs/ACs [Huber et al. (1991), incorporated by reference herein]; PA1 3. The cytokines and mediators of No. 1, above, induce the local endothelium to express P-selectin, the vascular cell adhesion molecule (VCAM-1), inducible cell adhesion molecule (INCAM-110), and von Willebrand's factor (vWF, Factor VIII antigen), which are reported binding determinants for GAGs/ACs [Bevilaqua et al. (1993); Bevilacqua et al.(1993)]; P-selectin is reported to bind GAGs [Bevilacqua et al.(1993)]; PA1 4. Integrins, including but not limited to VLA-4, are induced on circulating white blood cells, including lymphocytes, during various disease processes (see below); VLA-4 has a distinct binding site on the (induced) endothelial selectin, VCAM-1 (see No. 3, above); fibronectin, which is abundantly present in plasma and also available from tissue sites, has a distinct and separate binding site on VLA-4 [Elices et al. (1990)]; since fibronectin has specific binding sites for GAGs/ACs [Bevilaqua et al. (1993)], these amplification steps provide a strong additional mechanism for site localization of GAGs/ACs; PA1 5. The chemoattractants, MCP and IL-8, lymphocyte integrin, VLA-4, platelet factor, PAF, and coagulation factors, thrombin, fibronectin and others, diffuse from local tissue and blood, respectively, bind to the induced endothelial selections, and amplify adhesiveness and activation at the initial endothelial P-selectin sites for GAGs/ACs [Elices et al. (1990); Lorant et al. (1993)]; PA1 6. Tissue metalloproteinases become activated and expose new binding sites for GAGs/ACs in the tissues which underlie the activated endothelia. These new tissue binding sites include as follows [Ranney (1990); Ranney (1992); Travis (1993); Bevilaqua et al. (1993)]: PA1 7. White blood cells are attracted to the site, become activated and release additional proteolytic enzymes, thereby amplifying No. 6 and increasing the exposure of binding sites for GAGs/ACs in the tissue matrix. PA1 8. GAG/AC carriers selectively bind the induced and exposed determinants listed in Nos. 1-7, above, giving immune-type localization which is specific for induced binding sites (lectins) at the lectin-carbohydrate level characteristic of white-cell adhesion; PA1 9. In cases where the carrier substance has multivalent binding to the target binding substance, including for example, cases in which the carrier is an acidic oligosaccharide or polysaccharide or an acidic glycosaminoglycan, multivalent binding of the endothelial surface induces rapid extravasation of the carrier and bound active, and results in substantially increased loading of the underlying tissue matrix, relative to that achieved by antibodies, liposomes, and monovalent binding substances, such as hormones and monovalent-binding sugars; PA1 10. Adhesion of GAGs/ACs to induced and exposed tissue binding sites, reduces plasma backdiffusion of GAGs/ACs and their bound actives, thereby giving sustained retention within the tissue site; PA1 11. Controlled release of the diagnostic or drug activity from carriers comprising GAGs/ACs occurs gradually within the diseased site, thereby resulting in targeted controlled release; PA1 12. Tumor cells, microbial targets and damaged cellular targets within the tissue site, may selectively take up the GAG/AC plus bound diagnostic or drug active, based respectively, on: induced tumor anion transport pathways, microbial binding sites for GAGs/ACs, and proteolytically exposed cell-surface core proteins [Ranney Ser. Nos. 07/880,660, 07/803,595 and 07/642,033]--Fe uptake by hepatomas, Cr.sub.4 S uptake by prostatic adenocarcinomas; [Kjellen et al. (1977)] PA1 13. In cases where the carriers are hydrophilic or essentially completely hydrophilic, these carriers cause their bound actives to migrate (permeate) deeply into and throughout the tumor mass even at microanatomic sites distant from the tumor's typically irregularly spaced microvessels; and also to migrate out (permeate) into a small rim of normal tissue around each focus of disease, typically comprising a rim about 30-75 um thick; however, such carriers and/or their associated active substances (diagnostics or therapeutics) undergo selective uptake (internalization) by abnormal cells within tissue site and preferentially avoid uptake by normal cells within the site, thereby giving: PA1 14. In the case of hydrophilic carriers, including but not limited to GAGs/ACs, the non-targeted fraction of active is cleared rapidly and non-toxically, thereby minimizing: PA1 1. MCP: Experimental autoimmune encephalomyelitis in mice [Ransohoff et al. (1993)]; PA1 2. IL-8: Neovascularization: [Strieter et al. (1992)]; PA1 3. PAF: Reperfused ischemic heart [Montrucchio et al. (1993)]. PA1 1. ELAM-1: PA1 2. VCAM-1: PA1 3. INCAM-110: Chronic inflammatory diseases, including sarcoidosis [Rice et al. (1991)]. PA1 4. Integrin, beta 1 subunit cell adhesion receptor: inflammatory joint synovium [Nikkari et al. (1993)]. PA1 1. They are exceedingly expensive materials, available only by synthetic or semi-synthetic means, and hence, are not cost effective; PA1 2. They do not bind effectively at diseased sites under in vivo conditions, apparently due to the inability as monomeric binding substances to displace endogenous interfering substances which are pre-bound at these sites. PA1 1. GAGs allow a broader range of tumors and diseases to be targeted than that possible with antibodies (which typically target only a subset of histologic types--even within a given class of tumor, and hence, are typically ineffective from both a medical and cost/development standpoint); PA1 2. GAGs are projected to be effective over a greater time interval, from early onset of disease to progression and regression. PA1 1. Restrict initial biodistribution of the diagnostic/drug to the plasma compartment and thereby maximize the quantity of agent available for site targeting; PA1 2. Displace endogenous interfering substances which are pre-bound to diseased endothelium; PA1 3. Induce active endothelial translocation of the GAG-diagnostic/drug into the underlying tissue matrix; PA1 4. Afford rapid clearance and markedly reduced side effects of the attached actives.
Diagnostic and drug enhancement can be made to occur by a number of mechanisms, the principal ones being:
1. Effective TARGETING to tissue sites of disease; PA0 2. STABILIZATION during both storage and plasma transit; PA0 3. Prolonged RETENTION at the site of disease, giving a markedly increased area under the curve at the tissue site; PA0 4. RAPID CLEARANCE of the non-TARGETED fraction, thereby reducing background signal in imaging applications and reducing normal organ exposure and systemic toxicity in therapeutic applications.
Five further significant advantages of the present compositions and uses are:
Acidic or anionic saccharides and glycosaminoglycans have unique mechanisms of site localization and retention in vivo. They bind to the body's endothelial determinants which are selectively induced on the microvascular barrier by underlying tissue disease. Previous approaches to site targeting were directed at antigenic determinants. However, because these determinants are typically located in sequestered sites within the tissues, in other words at sites across the endothelial barrier and not within the bloodstream and not on its endothelial surface, carriers and agents injected into the bloodstream had no effective means to recognize and localize in the region of these target antigens. Stated another way, previous approaches ignored the major problem of inappropriate carrier distribution which resulted from its failure to recognize the vascular access codes required for efficient extravasation at disease sites. Hence, these carriers failed to effectively load the relevant tissue sites with effective concentrations of their bound actives.
Acidic or anionic saccharides, including glycosaminoglycans, dermatan sulfates and the new special dermatan sulfates, localize at target sites by binding first to complementary receptors on disease-site vascular endothelium, induce very rapid (ca. 3-minute) extravasation of the carrier and associated active agent, and then widely permeate throughout the underlying tissue matrix, forming a depot reservoir of the carrier-agent selectively at the site of disease (including tumors--even at sites up to several hundred micrometers distant from the typically irregularly spaced and perfused microvessels within the tumor matrix), and thirdly, bind to complementary receptors on the final target cells (including tumor cells), leading to induced tumor-cell internalization of GAG-actives (including DS-actives) (see Examples below). The new special class of dermatan sulfates (described just above and more extensively below) appears to perform this complementary binding function via their selectively enriched oversulfated saccharide sequences, which correlate with an enriched heparin cofactor II activity of at least about 220 U/mg, and which appear to bind the positively charged, cationic and/or structurally complementary receptors or lectins that are selectively induced on disease-site endothelium, tissue matrix and target cells (including in tumors). For the new dermatan sulfates, these binding and targeting functions and potencies occur without either the overall high sulfation/polysulfation or the incumbent toxicity and safety disadvantages thereof (as otherwise described herein).
The biological address of a disease site can be viewed in a fashion similar to that of a postal address, wherein effective carrier substances must (1) first recognize the "state" address of the signal endothelium induced by proximal tissue disease; (2) next extravasate and load the "city" address of the extracellular tissue matrix with locally effective doses of the diagnostic and therapeutic actives; and (3) finally bind and load the "street" address of the target cells and antigens. Previous approaches to site delivery have attempted to recognize the "street" address without first recognizing the "state" and "city" addresses.
The reason that acidic saccharide and sulfated glycosaminoglycan systems work substantially better than previous antigen-recognition approaches, is that they recognize the newly induced signals which the body uses to attract and target white blood cells into sites of tissue disease. When disease strikes at a local site, it initiates a cascade of local mediators within the tissue matrix and at the endothelial-blood interface which signal the blood cells and central body systems that inflammatory and immune cells are required within the tissue site. These mediators include cytokines, chemoattractants, cytotoxins, induced cell-surface adhesins, selectins and integrins, and various tissue-derived and blood-borne, soluble and cell-surface procoagulants. White cell accumulation begins within minutes and continues over days to weeks, depending on the nature, severity and persistence of local disease and the continued generation of tissue mediators and trans-endothelial signals.
As has now been reported and reviewed in detail [Ranney (1990); Ranney (1992); Bevilaqua et al. (1993); Bevilaqua et al. (1993); Travis (1993); Sharon et al. (1993), all incorporated herein by reference], tumors, infarcts, infections, inflammatory diseases, vascular disorders, and other focal diseases, characteristically induce the release of such host mediators, or cytokines, from resident macrophages and local tissue matrices. In certain diseases, alien mediators such as bacterial lipopolysaccharides (LPS), viral RNA, and tumor-derived inducers, including EMAP II, and chemoattractants may also be released. Although additional mediators remain to be elucidated, the principal ones have now been defined and include: interleukin 1 (IL-1), tumor necrosis factor (TNF), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor beta (TGF-beta), Lipopolysaccharide (LPS), single and double stranded nucleotides, various interferons, monocyte chemoattractant protein (MCP), interleukin 8 (IL-8), interleukin 3 (IL-3), interleukin 6 (IL-6), tumor-derived inducers and chemoattractant peptides (as above), various prostaglandins and thromboxanes. Certain ones of the preceding mediators induce the local generation and release of metalloproteinases, and these in turn, expose latent tissue binding sites, including intact and partially cleaved integrins, RDGS peptides, laminin, collagen, fibronectin, and cell-surface core-protein components of glycosaminoglycans.
Cytokines, including VEGF/VPF and monocyte chemoattractant protein (MCP); and tissue metalloproteinases and proteolytic tissue matrix fragments, directly induce the local endothelium to become adhesive for circulating white blood cells, including neutrophils, monocytes and lymphocytes. The induced endothelial adhesive molecules (adhesins) include: P-selectin (gmp-140), E-selectin (ELAM-1), intercellular cell adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), inducible cell adhesion molecule, (INCAM-110), von Willebrand's factor (vWF, Factor VIII antigen) (see below for disease states which activate these respective types of endothelial adhesins). Additional cascades become activated which indirectly amplify endothelial adhesiveness. These include (1) coagulation factors, especially fibronectin, tissue factor, thrombin, fibrinogen, fibrin, and their split products, especially fibronectin split products and fibrinopeptide A; (2) platelet-derived factors: platelet activating factor (PAF), glycoprotein IIb/IIIa complex; (3) white-cell (a) L-selectin, and (b) integrins, including VLA-4 (very late antigen 4); and (4) numerous complement factors.
The preceding pathologic processes and signals are involved, directly or indirectly as follows, in the binding and site localization of acidic carriers, including acidic saccharides (AC) and glycosaminoglycans (GAGs) (Note that in the following outline, potential tissue binding sites are designated as "GAGs" and "ACs").
a. fibronectin fragments; PA2 b. collagen fragments; PA2 c. laminin fragments; PA2 d. RGDS peptides; PA2 e. Exposed core proteins of GAGs; PA2 a. In cases of diagnostic imaging applications: very sharp definition of the boundary between tumors or infarcts and the surrounding normal tissues; PA2 b. In cases of therapeutic applications: PA2 a. in imaging uses, background signal intensity; PA2 b. in all uses: PA2 a. Liver portal tract endothelia in acute and chronic inflammation and allograft rejection [Steinhoff et al. (1993)]; PA2 b. Active inflammatory processes, including acute appendicitis [Rice et al. (1992)]. PA2 a. Simian AIDS encephalitis [Sasseville et al. (1992)]. PA2 b. Liver and pancreas allograft rejection [Bacchi et al. (1993)].
(1) protection against spread of disease at the rim; PA3 (2) relative protection of normal cells within and adjacent to the site of disease, from uptake of cytotoxic drugs. PA3 (1) normal organ exposure; and PA3 (2) systemic side effects.
Regarding the above outline, the tumor-selective GAG-binding cytokines, VEGF/VPF and MCP, are now known to be present in all three of the following microanatomic locations: tumor-cell surface, tumor extracellular matrix, and local tumor neovascular endothelium. Hence, these cytokines provide receptor targets for GAG-agents at all three of the key tumor sites: tumor endothelium, tumor extracellular matrix, and tumor cells proper. The presence of these cytokines selectively on tumor endothelium, allows for site-selective binding of intravascularly administered GAG-agents to tumor microvessels and very rapid (ca. 3-minute) selective extravasation of GAG-agents across the VEGF/VPF-"permeabilized" endothelium. Note: such "permeabilization" is recently shown to actually (a) comprise rapid transport by vesicular endosomes which are markedly enlarged (over the standard 120 nm Palade vesicles characterizing normal endothelium) and markedly increased in number (over normal vascular endothelium) [Senger et al. (1993), incorporated by reference herein]; and (b) comprise anatomically non-porous vascular endothelium, as assessed by macromolecular and particulate markers of true microfiltration porosity. The presence of VEGF/VPF and MCP cytokines on tumor cell surfaces may account for selective tumor-cell internalization of GAG-agents, as shown in certain of the Examples below. Importantly, the presence of these cytokines plus the GAG-binding peptides of No. 6 (above) in the large extracellular volumes of the tumor matrix, accounts in part, for the large tumor-tissue reservoirs of GAG-associated agents (including metal chelates) which are observed by MRI contrast enhancement (see Examples below). The relatively slow (ca. 7-hour) backdiffusion of such agents into the bloodstream, further corroborates the presence of such extracellular tissue-matrix receptors. Importantly, the combination of: (1) prolonged tumor retention of Gag-agents as an extracellular reservoir (depot); (b) tumor-cell internalization of a portion of this extracellular agent; and (c) very rapid blood and body clearance of the non-targeted portion, provides the following surprising and unexpected advantages for in vivo imaging (including MRI contrast enhancement) and therapy: (a) enhanced tumor selectivity; (b) prolonged, high "areas under the curve" (AUC's) in tumor; (c) short, low AUC's in blood; (d) minimization of local and systemic toxicities. Additionally, involving the above outline, the following (A) cytokines and mediators; and (B) selectins, integrins and adhesins are reported to be induced by various disease states in addition to that reported for tumors, above [Bevilaqua et al. (1993)]. Representative non-oncologic induction also occurs as follows.
A. Cytokines and mediators.
B. Selectins, Integrins and Adhesins.
It is apparent from the above, that broad categories and many specific types of focal tissue disease may be addressed by the carriers and actives of the present invention, both for diagnostic and therapeutic uses, including tumors, cardiovascular disease, inflammatory disease, bacterial and viral (AIDS) infections, central nervous system degenerative disorders, and allograft rejection. It will also be obvious to those skilled in the art, that numerous additional disease states may be selectively addressed by the carriers disclosed in this invention.
The site selectivity of glycosaminoglycans (GAGs) appears to mimic an immune mechanism at the level of white-cell targeting rather than antibody targeting. Because antibodies have extremely high specificities, they characteristically miss major subregions of disease foci (typically as great as 60% of tumor cells are nonbinding). Recently, one of the GAG-binding determinants of endothelial P-selectin has been identified as sialyl Lewis x. Others are in the process of identification. Notably, the available nonvalent oligosaccharides specific for sialyl Lewis x suffer from two critical problems:
There are two apparent benefits of the relatively broader range of GAG specificities and redundancy of GAG binding sites present on diseased endothelium, tissue matrix and cells:
Despite the broader targeting specificity of GAGs over antibodies, their favorable clearance and avoidance of uptake by normal cells reduce systemic and local toxicities, even though more than one type of disease site may undergo targeted accumulation of the diagnostic/drug within its extracellular matrix.
The polymeric and multivalent binding properties of GAGs both are very important for optimal site localization of the attached diagnostic/drug. GAG molecular weights of generally ca. 8,000 to 45,000 MW, preferably 10,000 to 23,000 MW and more preferably 13,000 to 19,000 MW, are important in order to: